Biased pollen transfer by bumblebees favors the paternity of virus-infected plants in cross-pollination

Summary We used a green fluorescent protein marker gene for paternity analysis to determine if virus infection affected male reproductive success of tomato in bumblebee-mediated cross-pollination under glasshouse conditions. We found that bumblebees that visited flowers of infected plants showed a strong preference to subsequently visit flowers of non-infected plants. The behavior of the bumblebees to move toward non-infected plants after pollinating virus-infected plants appears to explain the paternity data, which demonstrate a statistically significant ∼10-fold bias for fertilization of non-infected plants with pollen from infected parents. Thus, in the presence of bumblebee pollinators, CMV-infected plants exhibit enhanced male reproductive success.


INTRODUCTION
Cross-pollination, the transfer of pollen grains from the stamens of one plant to the stigma of another, has the potential to increase genetic diversity of the next generation. 1 Cross-pollination also contributes to gene flow between and within populations. Paradoxically, whilst gene flow can be an important agent of evolution, for example, by facilitating dissemination of a new trait, it can also act as an 'evolutionary glue' that limits genetic diversity within a species, preventing genetic drift. [2][3][4][5] A fuller understanding of the mechanisms shaping pollination is important, not only because of its role in gene flow, but also for practical reasons, particularly in the light of global pollinator decline. 6 Over three-quarters of major crops grown worldwide and 90% of wild flowering plants rely on animal-mediated pollination to set fruit and seed and, out of all animals, bees are the principal pollinators. [7][8][9] Much research has been devoted to understanding the inherent features of flowers that influence pollinator behavior (e.g., floral signals and rewards 1 or the trade-offs between attracting pollinators and deterring predation. 5 However, the effects of plant pathogen infection on pollen movement between plants have not been deeply explored. It was previously shown that volatile organic compounds emitted by virus-infected bean (Phaseolus vulgaris L.) and tomato (Solanum lycopersicum L.) plants attract bumblebees (Bombus terrestris). 10,11 When bumblebees visit tomato flowers, they vibrate to extract pollen ('buzz pollination') and, in doing so, maximize seed production by increasing the self-pollination rate. 11 Cucumber mosaic virus (CMV) infection decreases tomato seed yield, except in the presence of bumblebees whose buzz pollination activity rescues infected plant seed yields to levels equivalent to those of non-infected plants. 11 It was concluded that a greater preference of pollinators for flowers of virus-infected plants would increase their success as female parents, by increasing the probability of ovule fertilization. 11 In addition, it was inferred that for a wild population of plants evolving and that this could constitute a 'payback' from a virus to its susceptible hosts that would blunt selection pressure for resistance and would, as a byproduct, enable survival of alleles for susceptibility within a plant population. 11 infected, appears to explain the paternity data, which show a statistically significant $10-fold bias for fertilization of non-infected plants with pollen from infected parents. Thus, in the presence of bumblebee pollinators, CMV-infected plants exhibit enhanced male and female reproductive success.

Establishment of a paternity tracking system
Paternity experiments using naturally occurring DNA sequences and sequence variants as markers to investigate insect-mediated pollen transfer are useful for studies of gene flow in plants. 12,13 To investigate pollinator-mediated gene flow between plants we used transgenic tomato plants of the Moneymaker variety carrying the coding sequence for GFP under the control of the constitutive cauliflower mosaic virus 35S promoter. 14 Inheritance of this artificial marker gene was scorable visually because it conferred fluorescence on seed tissue as well as most other plant tissues, and it allows investigation of pollen transfer using plants that are genetically identical except for the possession, or not, of the GFP transgene ( Figure 1).
Expression of the GFP transgene in this tomato line (GFP#6) induces no significant changes in carbon metabolism 14 and is therefore unlikely to have altered bee-perceivable cues such as changes as emission of volatile organic compounds or floral pigments. Nevertheless, we checked that genetic modification had not affected seed yield or influenced in some way the buzz-pollination behavior of bumblebees. Fifty-plant arrays comprising 25GFP-transgenic and 25 non-transgenic plants arranged alternately were set up in a glasshouse flight arena with a bumblebee nest placed centrally ( Figure S1). To confirm that GFP transgene expression did not influence bumblebee buzz-pollination behavior, we measured the time bumblebees spent sonicating flowers, i.e., gripping the floral cone and vibrating to release pollen ( Figure S2A). Bees sonicated flowers of non-transgenic plants and GFP-transgenic plants for similar durations ( Figure S2A). Fruits arising from sonicated flowers (i.e., visited by bumblebees) and fruits that developed from unvisited flowers were harvested and the seeds collected and counted. For fruit produced by unvisited flowers, the number of seeds per fruit was similar for non-transgenic and GFP-transgenic plants ( Figure S2B). As expected from previous investigations, 11,15 buzz-pollination significantly increased seed production, and the enhancement of self-pollination was statistically indistinguishable between non-transgenic and GFPtransgenic plants ( Figure S2B). Thus, constitutive expression of the GFP transgene did not affect any tomato phenotype influencing reproduction or cues affecting bumblebee pollination behavior.  Figure 1). Fruit was collected from non-transgenic plants and seeds examined for expression of GFP using a low magnification epi-fluorescence microscope. Seeds resulting from self-pollination were not fluorescent, but seeds resulting from cross-pollination fluoresced because of the presence of GFP in the embryo, i.e., demonstrating that fertilization of the seed had resulted from bumblebee-mediated transfer of GFP-transgenic pollen to the flower of a non-transgenic plant (Figures 1 andS3). The cross-pollination rate between non-infected plants, that is, the basal rate of cross-pollination in the absence of virus infection, ranged from 0.01 to 0.1% in three independent experiments ( Figure 2A; Table S1). This low basal rate of cross-pollination is consistent with the predominantly autogamous (self-fertilizing) reproduction of tomato, and with previous estimates of natural  iScience Article cross-pollination in the field. For example, Groenewegen et al. 16 used a transcription factor gene controlling anthocyanin biosynthesis 17 as a paternal marker to detect crosses with a tomato line homozygous for the mutant allele and scored seedlings for production of anthocyanin. This approach yielded a mean value for the cross-pollination rate of 0.0721% when pollinators were present, 16 which is commensurate with our estimated rate of cross-pollination ( Figure 2A; Table S1).
The rates of bee-mediated pollen transfer from mock-inoculated to CMV-infected plants and from CMVinfected plants were compared to the basal cross-pollination rate ( Figure 2). Analysis by either binomial logistic regression ( Figure 2A) or probability ( Figure 2B) showed the proportion of pollen transferred from mock-inoculated plants to CMV-infected plants was not significantly elevated relative to the basal cross-pollination rate between healthy plants. However, pollen transfer from CMV-infected plants to mock plants was enhanced by an order of magnitude, relative to the basal cross-pollination rate ( Figure 2A). This remarkable observation indicates that CMV infection leads to a markedly increased probability of transfer of pollen from infected plants to non-infected plants, whereas having no significant impact on pollen movement in the opposite direction.
All GFP fluorescent seeds were viable. Seedling leaf tissue was used to carry out reverse transcriptioncoupled polymerase chain reactions (RT-PCRs) to detect expression of the transcript of the neomycin phosphotransferase II (NPTII) transgene. NPTII (conferring kanamycin resistance) is present in the transfer DNA containing the GFP marker gene. 14,18,19 RT-PCR showed that seeds showing GFP fluorescence also expressed NPTII, confirming that pollen from a GFP-transgenic tomato plant had been transferred by a bumblebee to the stigma of a non-transgenic plant ( Figure 3). Seeds resulting from cross-pollination germinated successfully, showing that neither cross-pollination nor CMV infection of the paternal parent plant affected the viability of the resulting seed. We found that progeny from crosses were not infected with CMV ( Figure 3). This is consistent with the previous literature on CMV, i.e., that this virus is not known to be transmitted via tomato pollen, and that bumblebees are not CMV vectors. 20,21 A selection of self-pollinated seeds from fruits harvested from healthy and CMV-infected plants had similarly high germination rates also indicating that seeds from virus-infected plants were as viable as those from healthy plants ( Figure S4).

Bumblebees spent less time interacting with flowers on CMV-infected plants
To understand how the bias for bumblebee-mediated pollen transfer from infected to non-infected plants occurs, we first examined if bumblebee foraging behavior differed between visits to CMVinfected and mock-inoculated plants. Previous work suggested that bumblebees may spend more time foraging on flowers from CMV-infected plants. 11 However, using bee tracker software to time flower visits of bees over the course of a 2-3h pollination experiment and by producing a larger dataset, we The left panel shows that some seeds (offspring) from non-transgenic parents occasionally showed GFP fluorescence (+) and also expressed NptII and that no offspring were infected with CMV (established by RT-PCR using appropriate primers, labeled CMV). This confirmed that fluorescent seeds were the result of cross-pollination. RT-PCR was performed on RNA extracted from parent plants and germinated offspring. cDNA was generated using random primers and subsequently used in PCR reactions using appropriate primers ( iScience Article found that bees consistently spent half as long visiting flowers on infected plants than they did visiting flowers on healthy plants ( Figure 4). This was largely owing to bees paying fewer visits to virus-infected plants ( Figure S5).
Could the amount of pollen released from virus-infected plants as a reward to foraging bumblebees be lower than that from non-infected plants and therefore account for the bees' fewer visits to virus-infected plants?
Although the pollen of CMV-infected plants is as viable as that from non-infected plants, 11 we wondered if pollen maturation or the ease with which it would be released by sonication would be affected on CMV-infected plants. Our rationale for this hypothesis was that the CMV 2b protein is known to inhibit expression of c. 90% of plant genes regulated by the phytohormone jasmonic acid (JA) 23 and because, among other things, JA is a key factor controlling pollen development in many plants, although in tomato it appears to be less critical. 24 Pollen was released by mechanical sonication of flowers and weighed using a microbalance.  iScience Article No significant difference in total pollen mass per flower was detected ( Figure 5A). But although CMV infection of tomato did not decrease the overall quantity of pollen produced, it did affect the timing of peak pollen availability ( Figure 5B). We found that although flowers of non-infected plants released peak amounts of pollen over three days (R10 4 grains per 5 s vibration), flowers of CMV-infected plants released most of their pollen over a two-day period ( Figure 5B). Although it is unclear how this change in timing would explain the bias for pollen transport from CMV-infected to uninfected plants, it may explain why, overall, bees spend less time interacting with flowers on CMV-infected plants (Figures 5 andS5). iScience Article been proposed as being inversely proportional to gene flow mediated by by cross-pollination. 25,26 That is, gene flow via cross-pollination is inhibited if pollinators remain on the same plant or if they subsequently visit only plants of the same genotype. In contrast, gene flow is favored when pollinators migrate to forage on plants of the same species but of differing genotype. The greatest chance of a cross-pollination event occurring will be when two flowers are visited in immediate succession. 25,26 We set up experiments to track bumblebee movement between flowers of mock-inoculated and CMVinfected plants to find out if the cross-pollination bias (Figure 2) was explainable by patterns of pollinator foraging behavior, i.e., did the likelihood of pollinator residence differ for infected versus non-infected plants? The patterns of movement between flowers for individual bumblebees were recorded in arrays of 3 x 10 plants. These arrays comprised alternating CMV-infected and mock-inoculated tomato plants ( Figure S6). A bumblebee under these conditions, following its initial visit to a flower, had two foraging choices available. First, it might move to a plant of the same treatment group (from a mock-inoculated plant to another mock-inoculated plant, or from a CMV-infected plant to another CMV-infected plant), and this would be 'residence' in the modeling terminology of Cresswell et al. 25 Alternatively, the bumblebee might move to a plant of the other treatment group, i.e., from a CMV-infected plant to a mock-inoculated plant or from a mock-inoculated plant to a CMV-infected plant. If bumblebees move in an unbiased fashion from flower to flower, an even split between choices would have been expected. Initial choices of individual bumblebees for their initial foraging bout were unbiased between CMV-infected and mock-inoculated plants. However, it was found consistently that the rarest subsequent move by a bumblebee was from a virus-infected plant to another virus-infected plant, and that bumblebees that alighted first on flowers of mock-inoculated plants showed the highest degree of residency, iScience Article that is, choosing to visit another mock-inoculated plant ( Figure 6, Table S2). Thus, CMV infection of tomato affected bumblebee-flower interactions in that it was more likely that a bumblebee would visit a flower on a mock-inoculated plant after buzz-pollinating a flower on an infected plant. In the light of previous analyses of gene flow in plant-pollinator systems, 25,26 this provides a mechanism to explain why transfer of pollen carrying a GFP marker gene is more probable when the donor (i.e., the male parent) is infected with CMV ( Figure 2).

Conclusions
Pollen from flowers of CMV-infected plants was more likely to be transferred by bumblebees to the stigmas of non-infected plants. This bias may be caused by distasteful substances accumulating in the pollen or differences in, for example, the tactile cues of the flower, or by other factors to be investigated in future work. These results may explain the increased success of infected plants as male parents in cross-pollination (Figure 2), although they do not throw light on the mechanism by which female reproduction (seed production) of CMV-infected plants is rescued by bumblebee-mediated self-pollination. 11 Olfactometry experiments have shown that bumblebees are attracted to the VOCs emitted by virus-infected plants. 10,11 Under the conditions used in the present study it does not appear that CMV-induced changes in VOC emission influenced the initial choices of bumblebees to settle on infected or noninfected plants. We can speculate that VOCs may play some role in enhancing bee visitation to CMV-infected plants, for example, by compensating for the decreased salience of infected plants, which are markedly shorter than mock-inoculated plants ( Figure S7) or, paradoxically given their attractiveness in olfactometry experiments, VOCs may provide a cue for bumblebees that had already visited an infected plant to avoid visiting another infected plant (Figures 4 and 6). Our main conclusion is that gene flow via pollen is increased in tomato when plants are infected with CMV. This increased male reproductive success, combined with the rescue of seed production for infected plants when pollinators are present, 11 will counteract the negative effects of viral infection on reproductive fitness, thus providing a 'payback' to susceptible host plants, which is not available to resistant hosts. As mentioned earlier, under natural conditions gene flow may aid evolution, but it can also counter genetic drift in an interbreeding population and promote genetic homogeneity. [2][3][4][5] We suspect that virus-induced gene flow will tend to favor homogenization. Thus, if viruses enhance the paternity of wild plants under natural conditions, a by-product of increased gene flow from infected plants would be increased spread of host genes from the infected plant, among which will be those encoding factors that support virus infection. This idea is consistent with a hypothesis put forward by Groen et al. 11 that for wild plants under natural conditions such a payback would promote the dissemination of alleles for susceptibility and would counteract the selective advantage of alleles for resistance. Once established, such a situation would be advantageous both for the virus and its susceptible plant hosts.

Limitations of the study
Our study was carried out in a glasshouse and although this approach provides robust data on the influence of plant virus infection on bumblebee-mediated cross-pollination, bumblebee interactions will be more complex in the wild environment, where environmental and other biotic factors may influence pollinatorplant interactions.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following: CMV isolate PV0187 (Subgroup IA) was obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ, www.dsmz.de). 11 Virions were purified from Nicotiana benthamiana 3 weeks post-inoculation and stored for up to 3monthsat 4 C. 27 Seedlings were mechanically inoculated on both fully emerged cotyledons after one week of growth by dusting with Carborundum (silicon carbide) powder and gently rubbing 10 mL of purified CMV virions (100 mg mL À1 suspension in sterile water) with a frosted glass microscope slide over the cotyledon surface. Mock-inoculated plants underwent the same procedure with sterile water.
At two weeks of age, inoculated and mock-inoculated plants were transplanted into 12 3 12 3 20 cm pots and grown for another two weeks under controlled growth conditions before transfer to the University of Cambridge Botanic Garden glasshouse. Plants were maintained at 15-25 C, and Lucalux LU 400 W/PSL lights were automatically activated between the hours of 0400 and 2000 when daylight levels fell below 150 W/m 2 . Humidity was approximately 55%. Tomato plants were placed randomly within the glasshouse space available (to minimize the potential environmental gradient effects such as light or humidity) and after beginning to flower (3-6 weeks later) used in pollination experiments.  Figure S1D). To measure pollen transmission from mock-inoculated to virus-infected plants, the reciprocal experiment was carried out, i.e. mock-inoculated GFP-transgenic and CMV-infected non-transgenic tomato plants were used. Pollen transmission rates were also measured between mock-inoculated GFP-transgenic and non-transgenic tomato plants. When bumblebees had visited all the plants at least once, the experiment was concluded and all buzz-pollinated flowers (identified from characteristic wounding marks left by bees after buzz-pollination; Figures S1D-S1F) were labeled with a jeweler's tag ( Figure S1G). Fruits arising from buzz-pollinated and non-pollinated flowers were allowed to develop on these plants and harvested (8-12 weeks later). The seed from individual fruit was collected by rinsing through a sieve.

Cross-pollination experiments
To determine seed paternity, the presence of marker genes in tomato seeds was ascertained by epi-fluorescent microscopy (M165 FC Fluorescence Dissecting Stereomicroscope equipped with a GFP filter, Leica Microsystems, Milton Keynes, UK) for the GFP protein to ascertain the cross-pollination rate. GFP fluorescing progeny seed harvested from non-transformed tomato plants were indicative of a cross-pollination event ( Figure S3). These seeds were germinated, and RNA was extracted from cotyledons using Norgen Total RNA Purification Plus Kit (Norgen Biotek, Thorold, ON, Canada) and used in RT-PCR for NPTII (kanamycin resistance gene) as further confirmation that these seeds were progeny of a GFP-transgenic pollen donor and a non-transgenic pollen recipient (primers used: Mechanical release and collection of pollen As described by Groen and colleagues, 11 artificial buzz-pollination was carried out using an electrically actuated toothbrush ('Oral-B': Proctor and Gamble, Cincinnati, USA). The brush was covered with a finger from a disposable glove, which was changed between flowers.  28 to when they collapsed. The sum of all pollen yields from a single plant was divided by the number of flowers sonicated, to give a mean mass per flower.
To evaluate the pollen yield per day from open (stage 6) tomato flowers, pollen released from 5 s of sonication was collected into a single microfuge tube every day from when flowers first opened to when they collapsed. Pollen released from each flower per day was suspended in 200 mL of water and counted in technical triplicates using a cell-counting chamber under a microscope.

QUANTIFICATION AND STATISTICAL ANALYSIS
Data from cross pollination experiments shown in Figure 3 were analyzed using binomial logistic regression. Data from the i th experiment consists of a count of the number of GFP labeled seeds, s i , together with a count of the total number of seeds collected in the experiment as a whole, N i . We fitted the following model to these data, modeling p i the probability in the i th experiment of the pollen donor being a GFP labeled plant in whicha MM is the fitted logarithmic odds ratio in the mock-mock experiments, a MV and a MV are treatment effects for mock-virus and virus-mock experiments, respectively, and the indicator function c MV ðiÞ and c VM ðiÞ reflect the treatment in the i th experiment. Statistical differences between any pair of treatments was tested by comparing the fit of this model with the simpler nested model in whicha MV = a VM = 0 by way of a likelihood ratio test. Differences between individual treatments were then assessed by checking whether the 95% confidence intervals on the fitted treatment effects a MV and a VM overlapped 0 (and/or each other).

ADDITIONAL RESOURCES 'BeeTracker'
BeeTracker is a small GUI (graphical user interface) program, developed with C++ and Qt, which eases the tracking of pollinator visits to plants on a small scale (for example in behavioral arenas, cages or glasshouses), particularly when tracking the movements of multiple pollinators. BeeTracker shows a grid of configurable size, which the user matches to the experiment layout. The cells of the grid are clicked to start the timing of visit, and clicked again to indicate the visit has ended. Multiple visits can be tracked at a single location simultaneously. When the program is closed (at completion of experiment), data is automatically saved as a file containing all the recorded visits in tab-delimited columns for analysis. Access to BeeTracker code is at https://github.com/elderfd/BeeTracker.